A variety of Clostridium sp. strains that secrete neurotoxins have been discovered since 1890s, and the characterization of toxins that are secreted from these strains has been made for the past 70 years (Schant, E. J. et al., Microbiol. Rev., 56:80, 1992).
Neurotoxins derived from the Clostridium sp. strains, that is, botulinum toxins, are classified into seven types (types A to G) depending on their serological properties. Each of the toxins has a toxin protein having a size of about 150 KDa and naturally contains a complex of several non-toxic proteins. A medium complex (300 kDa) is composed of a toxin protein and a non-toxic non-hemagglutinin protein, and a large complex (450 kDa) and a very large complex (900 kDa) are composed of the medium complex bound to hemagglutinin (Sugiyama, H., Microbiol. Rev., 44: 419, 1980). Such non-toxic hemagglutinin proteins are known to function to protect the toxin from low pH and various proteases in the intestines.
The toxin is synthesized as a single polypeptide having a molecular weight of about 150 kDa in cells, and then cleaved at a position of ⅓ starting from the N-terminal end by the action of intracellular protease or treatment with an artificial enzyme such as trypsin into two units: a light chain (L; molecular weight: 50 kDa) and a heavy chain (H; molecular weight: 100 kDa). The cleaved toxin has greatly increased toxicity compared to the single polypeptide. The two units are linked to each other by a disulfide bond and have different functions. The heavy chain binds to a receptor of a target cell (Park. M. K., et al., FEMS Microbiol. Lett., 72:243, 1990) and functions to interact with a biomembrane at low pH (pH 4) to form a channel (Mantecucco, C. et al., TIBS., 18:324, 1993), and the light chain has pharmacological activity, and thus imparts permeability to cells using a detergent or interferes with the secretion of a neurotransmitter when introduced into cells by, for example, electroporation (Poulain, B. et al., Proc. Natl. Acad. Sci. USA., 85:4090, 1988).
The toxin inhibits the exocytosis of acetylcholine at the cholinergic presynapse of a neuromuscular junction to cause asthenia. It has been considered that treatment with a very small amount of the toxin exhibits toxicity, suggesting that the toxin has any enzymatic activity (Simpson, L. L. et al., Ann. Rev. Pharmaeol. Toxicol., 26:427, 1986).
According to a recent report, the toxin has metallopeptidase activity, and its substrate is composed of synaptobrevin, syntaxin, a synaptosomal associated protein of 25 KDa (SNAP25) or the like, which are the unit proteins of an exocytosis machinery complex. Each type of toxin uses one of the above-described three proteins as its substrate, and it is known that type B, D, F and G toxins cleave synaptobrevin at a specific site, type A and E toxins cleave SNAP25 at a specific site, and type C cleaves syntaxin at a specific site (Binz, T. et al., J. Biol. Chem., 265:9153, 1994).
Particularly, type A botulinum toxin is known to be soluble in a dilute aqueous solution at a pH of 4.0-6.8. It is known that a stable non-toxic protein is separated from neurotoxin at a pH of about 7 or higher, and as a result, the toxicity is gradually lost. Particularly, it is known that the toxicity decreases as pH and temperature increase.
The botulinum toxin is fatal to the human body even in small amounts and is easy to produce in large amounts. Thus, it constitutes four major bio-terror weapons together with Bacillus anthracis, Yersinia pestis and smallpox virus. However, it was found that, when type A botulinum toxin is injected at a dose that does not systematically affect the human body, it can paralyze local muscle in the injected site. Based on this characteristic, type A botulinum toxin can be used in a wide range of applications, including wrinkle removing agents, agents for treating spastic hemiplegia and cerebral palsy, etc. Thus, the demand for type A botulinum toxin has increased, and studies on methods of producing botulinum toxin so as to satisfy the demand have been actively conducted.
A current typical commercial product is BOTOX® (a purified neurotoxin complex of type A botulinum toxin) that is commercially available from Allergan, Inc., USA. A 100-unit vial of BOTOX® is composed of about 5 ng of a purified neurotoxin complex of type A botulinum toxin, 0.5 mg of human serum albumin and 0.9 mg of sodium chloride and is reconstituted using sterile saline without a preservative (injection of 0.9% sodium chloride). Other commercial products include Dysport® (a complex of Clostridium botulinum type A toxin and hemagglutinin, which has lactose and human serum albumin in a pharmaceutical composition containing botulinum toxin and is reconstituted using 0.9% sodium chloride before use) that is commercially available from Ipsen Ltd., UK, MyoBloc™ (an injectable solution (a pH of about 5.6) comprising botulinum type B toxin, human serum albumin, sodium succinate and sodium chloride) that is commercially available from Solstice Neurosciences, Inc. Conventional methods used to produce botulinum toxins include an acid precipitation method, a precipitation method by salt, and a chromatographic method.
For example, Japanese Patent Laid-Open Publication No. 1994-192296 discloses a method of producing a crystalline botulinum type A toxin by culturing Clostridium botulinum bacteria, followed by acid precipitation, extraction, addition of nuclease, and crystallization. Further, U.S. Pat. No. 5,696,077 discloses a method of a crystalline botulinum type B toxin by culturing Clostridium botulinum bacteria, followed by acid precipitation, extraction, ion exchange chromatography, gel filtration chromatography and crystallization.
Simpson et al. produced a botulinum type A toxin by purifying botulinum neurotoxin by gravity flow chromatography, followed by HPLC, capture using affinity resin, size exclusion chromatography, and ion (anion and cation) exchange chromatography including the use of two different ion exchange columns (Method in Enzymology, 165:76, 1988), and Wang et al. used precipitation and ion chromatography to purify a botulinum type A toxin (Dermatol Las Faci Cosm Surg., 2002:58, 2002).
Moreover, U.S. Pat. No. 6,818,409 discloses the use of ion exchange and lactose columns to purify a botulinum toxin, and U.S. Pat. No. 7,452,697 discloses a botulinum type A toxin by ion exchange chromatography and hydrophobic chromatography. Korean Patent Laid-Open Publication No. 2009-0091501 discloses a method of purifying a botulinum toxin by acid precipitation and anion exchange chromatography, and US Publication No. 2013-0156756 discloses a method of purifying a botulinum toxin by anion exchange chromatography and cation exchange chromatography.
However, the conventional methods have problems in that the use of anion exchange chromatography adversely affects the gel banding pattern of botulinum toxins (U.S. Pat. No. 7,452,697) and in that these conventional methods are difficult to apply commercially, due to a long purification time. In addition, because Clostridium botulinum that is a botulinum toxin-producing strain is an anaerobic bacterium, there is a problem in that fermentation of the bacterium should be performed in an anaerobic system, and thus it is difficult to produce botulinum toxins in large amounts. In addition, there is a problem in that the active ingredient botulinum toxin purified by the above-described purification method is not clearly separated and identified, and thus contains impurities. Additionally, the conventional methods for production of botulinum toxins have a problem in that a filtration or dialysis process is necessarily performed to purify a high-purity botulinum toxin, suggesting that the purification process is complex and difficult.
Accordingly, the present inventors have made extensive efforts to solve the above-described problems occurring in the prior art, and as a result, have found that when a culture of a botulinum toxin-producing strain is treated with acid to form a botulinum toxin precipitate and the formed precipitate is purified by anion exchange chromatography, the steps of filtration, dialysis and ethanol precipitation can be omitted, and the process for production of the botulinum toxin is very easy, and a botulinum toxin having a purity of 98% or higher can be produced by this production method, thereby completing the present invention.